Since a cell's DNA is frequently damaged by both external and internal agents, it is critical that such damage be accurately repaired. Damage that is not repaired, or that is repaired incorrectly, can result in mutation and genetic instability. In some cases, such alterations can lead to the development of cancer. Common fragile sites are one type of genomic element that may contribute to the development of cancer. There are 76 common fragile sites in the human genome and although these sites are normally stable, they are frequently broken and stimulate crossovers in cells that lack adequate amounts of DNA polymerase. Many types of tumors have been shown to contain deletions, amplifications, or translocations at fragile sites, therefore an understanding of the mechanisms contributing to this instability can aid understanding of cancer progression and prognosis. A naturally-occurring fragile site has been identified in the yeast Saccharomyces cerevisiae, named fragile site 2 (FS2), that mimics the type of genetic instability observed at human common fragile sites. This fragile site is a hotspot for double-strand breaks (DSBs) and translocations in cells with low levels of DNA polymerase. This proposal will use this simple eukaryote model system to build on and extend this work. The specific aims of the proposal are to: 1) Investigate the mechanism of DNA double-strand break formation at S. cerevisiae FS2, 2) investigate the stimulation of reciprocal mitotic crossovers at S. cerevisiae FS2, and 3) Investigate DNA double-strand break formation and crossover stimulation at human fragile site sequences using the yeast model system. These studies will help us understand which chromosome structures and sequences are intrinsically unstable, and what cellular pathways have a role in generating or repairing DSBs at such sites. This knowledge will help elucidate the mechanisms involved in genetic instability in tumor cells and further understand the genetic alterations contributing to the progression of cancer in humans. PUBLIC HEALTH RELEVANCE: Cancer cells have a high level of genetic instability, including large chromosome abnormalities such as rearrangements, amplifications, and deletions, and small changes such as the mutation of single bases. Naturally-occurring "fragile sites" on human chromosomes are correlated with genetic instability in cancer cells, but it is not well understood how and why these fragile sites generate instability. This proposal investigates instability at fragile sites from both yeast and human cells and will help elucidate some of the sequences, mechanisms, and proteins that have a role in genetic instability at these sites, which will add to the understanding of tumor development.